Malaria is the major vector-borne parasitic disease for much of the developing world. Because of parasite and mosquito resistance to drugs and pesticides, new control strategies are needed. The long-term objective of the research is to understand the physiology, biochemistry, and genetics of mosquitoes that are resistant to malaria infections. This may suggest ways to replace natural populations with genetically transformed nonvector mosquitoes. Although there are many reports of incompatibility between mosquitoes and malaria parasites, very few systems have been characterized in detail. One exception is a specific type of resistance found in a genetically-selected strain of Anopheles gambiae wherein mosquitoes melanotically encapsulate ookinetes of many malaria species. A fully susceptible strain was also selected from the same parental colony. The proposed research will focus on identifying and examining candidate resistance genes from this system. Six serine proteases have been identified that map to one of the major resistance loci, the 2La chromosomal inversion. These proteases are evenly distributed across the inversion and will be very useful in high-resolution mapping of resistance by RFLP analysis. Pooled segregant analysis will also be used to generate additional markers. Following mapping, cosmid libraries will be produced and chromosomal walk begun. Preliminary investigations also suggest that at least one of the serine proteases to be used for mapping may be directly involved in mosquito immune responses. Transcription of this protease is induced 18-24 hrs after septic wounding an it is 29% identical to a horseshoe crab serine protease that is activated by fungal cell wall components. Further characterization of this and the other five serine proteases will proceed by analysis of Northern blots of malaria-infected and uninfected mosquitoes, as well as of mosquitoes inoculated with fungal or bacterial cell wall products. The function of the candidates judged most likely to participate in melanotic encapsulation will be further analyzed by two methods. First, fusion proteins will be used to produce antibodies for immunocytochemical studies of the spatial and temporal distribution of proteins in susceptible and resistant mosquitoes. Second, a Sindbis virus construct designed to enhance or knockout function will be introduced into mosquitoes followed by bioassay of ookinete encapsulation.